Antimicrobial Activity of Peganum Harmala L. on Streptococcus mutans Compared to 0.2% Chlorhexidine.

STATEMENT OF THE PROBLEM
Dental caries is one the most prevalent diseases that affects humans throughout their lives. Streptococcus mutans (S. mutans) is recognized as the most important microorganism during tooth cariogenicity. Reducing this germ in oral cavity can reduce the rate of tooth decays in humans.


PURPOSE
The present study compared the antimicrobial activity of ethanolic extract of Peganum harmala L. seeds and 0.2% chlorhexidine on S. mutans.


MATERIALS AND METHOD
Agar diffusion technique and micro broth dilution method were employed to test the antimicrobial effects of these two agents on S. mutans. Moreover, the cytotoxicity of ethanolic extract of P. harmala was studied on Vero cells by MTT (thiazolyl blue tetrazolium dye) colorimetric method. The data were analyzed with descriptive methods.


RESULTS
Concentrations of 50, 25, and 12.5 mg/mL of the extract made inhibition zones of bacterial growth around the wells; but, lower concentrations could not inhibit the growth of S. mutans. Besides, the antimicrobial effect of 0.2% chlorhexidine was more than 50 mg/mL of the extract. Minimum inhibitory concentration (MIC) of the extract on S. mutans was 1.83±0.6 mg/mL and minimum bactericidal concentration (MBC) was 4.3±1 mg/mL. The MIC and MBC for 0.2% chlorhexidine were reported to be 0.19 mg/mL, and 0.78 mg/mL, respectively. The extract concentrations more than 0.5 mg/mL were toxic and caused more than 50% Vero cell death.


CONCLUSION
Despite the remarkable antimicrobial effects of high concentrations of P. harmala on S. mutans, high cell toxicity of this plant would restrict its in vivo therapeutic use.


Introduction
Dental caries is one of the most common diseases that humans are afflicted with throughout their lives. As the incidence of dental caries is increasing in developing countries, it is very essential to apply new methods to decrease this disease. [1] Streptococcus mutans (S. mutans) is one of the most important factors that lead to caries formation. [2] S. mutans together with other mi-croorganisms produce different glucans and acidificate dental plaque. As a result, the calcified structure of tooth is destroyed and dental caries would occur. Currently, chemical elimination of dental plaque by using disinfectants and mouthwashes to help the mechanical methods is worthy. [3] Many plants defend themselves against herbivores, microorganism, and insects by producing some secondary metabolites. The antimicrobial effects of these herbal products can be used for many remedial purposes. [4] Peganum harmala L. (P. harmala) is a small genus of zygophylacess family which widely grows in steppe areas and sandy soils in Asia, Middle East, North Africa, Australia and America. [5] P. harmala is called 'Espand' in Iran and the seeds are burned to disinfect the environment. [4] Different pharmacological and therapeutic effects of P. harmala are investigated in many studies. [4,[6][7][8] The important remedial aspects of this herb are its as antitumoral, antibacterial, antifungal, anti-parasitic, antinociceptive, anti-inflammation, vaso-relaxant , and antispasmodic activities. This plant is also used for diabetes, jaundice, asthma, dermatitis and many other illnesses. harmala are beta-carbonyl derivations such as harmalin, harmalol, peganine, isopeganine, deoxyisopeganine; as well as quinazoline derivations such as vasicinone, vasicine, and deoxyvasicinone. [5,9] Most alkaloids of this herb are derived from the seeds and roots. Harmalin is the best-known alkaloid in several researches that was studied. [5,10] The ability to intercalate DNA and the resulting frame shift mutations are among the etiological factors for antibacterial effects of this plant. [11] Ethanolic extract of P. harmala can restrain the growth of Streptococcus pyogenes, Staphylococcus aureus and Staphylococcus epidermidis. [4] Additionally, the alcoholic and aqueous extract of P. harmala can reduce the growth of lactobacillus and Candida albicans, two common microorganisms in oral cavity. [12] Moreover, Bacillus subtilis and Proteus vulgaris are the most sensitive bacteria to the metabolites derived from this plant. [13] Chlorhexidine is the most effective mouthwash, used to reduce dental plaque germs. It has bacteriostatic and bactericidal effects with suitable durability in oral cavity. It is more effective on S. mutans than lactobacillus; however, several side effects on tooth and oral mucosa limit the long-term usage of this mouthwash. [14] Tooth discoloration, changing in taste, increasing supragingival calculus, allergy, and oral lesions are most common side effects of chlorhexidine. Tooth discoloration is the most common consumer complaint. [14][15] It seems essential to investigate other antimicrobial agents that have inhibitory effect on relevant microorganisms in oral and dental diseases with fewer side effects compared to chlorhexidine. This study aimed to investigate the antimicrobial effect of ethanol extract of P. harmala on S. mutans. In addition, the cytotoxicity of this extract was evaluated on Vero cells.

Materials and Method
This experimental study was done in Department of bovine serum was added to each well. Six wells containing medium without extract were considered as control. The plate was incubated at 37°C in 5% CO2 incubator for 72 hours. Having washed the wells with PBS, 50µL of MTT (thiazolyl blue tetrazolium dye) was added to each of them and they were incubated for 2 hours.
Next, 50 µL of dimethyl sulfoxide (DMSO) was set up in each well and incubated again for 30 minutes. The absorbance was measured, using a microplate reader at 405-450 nm. Absorbance values that were lower than the controls indicated reduction in cell growth. The concentration required to reduce the cell growth to less than 50% (CC50) was determined by using regression chart.

Results
The anti-bacterial effects of 3.125-50 mg/mL concentrations of ethanolic extract of P. harmala and 0.2% chlorhexidine were checked out by using well diffusion technique ( Figure 1).  (Table 1). method, all concentrations of more than 0.5 mg/mL of the extract caused cell death over 50% (Figure 2). In a study, the alcoholic and aqueous extract of P.
harmala seeds had a limiting effect on lactobacillus, as one of the important microorganisms in making dental caries. In this study, ethanol extract of the herb had a greater inhibitory effect than the aqueous extract. [4] Darabpour et al. [4] reported that the alcoholic ex- Corresponding studies are suggested to investigate the cytotoxicity of this herb on oral epithelial cells for more accurate results. Moreover, controlled in vivo studies seem essential to be conducted on the use of this extract per se or in combined preparations as mouth wash.

Conclusion
In